Reliable Protein-Protein Interaction Results, Fast and Easy. With fewer false positives than other Yeast 2 Hybrid Technologies

A key part of gene functional analysis and potential drug target discovery is an understanding of how proteins interact within the cell. The following products facilitate the characterization of these interactions in yeast systems.

Using the ProQuest™ Two-Hybrid System with Gateway® Technology provides the ability to quickly and easily move sequences of interest between the ProQuest™ System and other expression and analysis tools using easy recombinational cloning.

The ProQuest™ Two-Hybrid System utilizes MaV203 Competent Yeast Cells. A single copy of each of three GAL4 inducible reporter genes (HIS3, URA3, and lacZ) has been integrated into the yeast genome, providing four phenotypes, for easier identification of true interactors. The MaV203 host yeast strain also features low-copy (ARS/CEN) vectors for reduced toxicity and consistent expression levels for more reproducible.

By utilizing three reporter genes and low copy number vectors, the ProQuest™ System results in fewer false positives than other systems. A set of five control yeast strains that demonstrate phenotypes for determining the strength of interaction is also provided.

Bait and Prey

The ProQuest™ Two Hybrid System with Gateway® Technology provides specially engineered components for bait and prey expression.

The pDEST™ 32 bait vector features:
  • ARS-CEN sequence for low-copy number maintenance in yeast
  • attR sites for Gateway® cloning
  • LEU2 for selection of recombinants on medium lacking leucine

pDBleu, the supercoiled parent of pDEST™32 is included with the system to test potential interactors.

The pDEST™22 prey vector features:

  •  ARS-CEN sequence for low-copy number maintenance in yeast
  • attR sites for Gateway® cloning
  • TRP1 for selection of recombinants on medium lacking tryptophan

pEXP-AD502 for construction of a cDNA or genomic prey is also included with the kit.


Figure 1. Outline of procedure for ProQuest™ Two-Hybrid System with Gateway® Technology
Pre-made ProQuest™ Libraries are available for use in identifying putative protein-protein interactions in the ProQuest™ System. Libraries are available in the Gateway® vector pEXP-AD502 and in the standard vector pPC86. Use of the Gateway® vector allows you to shuttle sequences of interest into a wide variety of expression and analysis vectors using recombinational cloning. Pre-made ProQuest™ Libraries offer the following features to ensure the most complete representation of full-length clones:

  • First strand synthesis catalyzed by SuperscriptÞ II RNase H- Reverse Transcriptase
  • Large average insert size, typically 1.3 to 2.3 kb
  • Greater than 5 x 109 derived from >5 x 106 primary clones to ensure the presence of rare sequences

Libraries are directionally cloned downstream of the GAL4 activation domain to generate GAL4 AD-cDNA fusion proteins. Libraries may be screened for interacting proteins using the ProQuest Two-Hybrid System, conventional PCR, or plate screening procedures; further amplified; or used for in vivo production of single stranded plasmid DNA.